Hacat

The cell lines HaCat, U-937, HL-60, and MCF-7 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). U-937 and HL-60 cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, New York, NY, USA) (Euroclone, Pero, Italy), while HaCat and MCF-7 in Dulbecco’s Modified Eagle Medium (DMEM) were grown at 37 °C in 5% CO₂ atmosphere, then supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% l-glutamine, 1% ampicillin or streptomycin, and 0.1% gentamicin. All cell lines were treated with extract of CWND, CBND, and WND at different concentrations ranging from 1.25 mg/mL to 5 mg/mL from 24 h to 72 h. For the biological assays, N. damascena extracts were resuspended in dimethyl sulfoxide (DMSO) and used at the final concentrations indicated in each experiment. The DMSO was used as a negative control.

The six cell lines, A375, H460, HT29, MCF7, HepG2, and HaCaT (Lonza, Verviers, Belgium), were kept in culture and expanded at 37 °C in a humidified atmosphere of 5% CO₂ in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Basel, Switzerland) culture medium for HaCaT, HepG2, HT29, MCF7, and A375, and RPMI-1640 for H460, supplemented with 10% FBS, Penicillin/Streptomycin 100× (Euroclone, Devon, UK), Glutamax 100× (Invitrogen, Carlsbad, CA, USA) and non-essential amino acids 100× (Invitrogen). Phosphate buffer (PBS, phosphate-buffered saline, Ca²⁺ and Mg²⁺ free) and trypsin (Ca²⁺ and Mg²⁺ free) were supplied by Euroclone. The cells were plated 15 × 10³ perwell in 96-well tissue culture plates and allowed to attach for 24 h. Then, cells were treated with PECS dissolved in culture medium supplemented with 1% FBS at different concentrations for 48 h (50 μg/mL, 80 μg/mL, 110 μg/mL, 140 μg/mL, 170 μg/mL, and 200 μg/mL). PECS were dissolved in sterile H₂O at 100 mg/mL.